Macrocyclic polypeptides

ABSTRACT

Disclosed herein are macrocyclic polypeptides having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-2.37 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids, libraries of such polypeptides, and uses thereof.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No. 16/610,574, filed Nov. 4, 2019, which is a U.S. national phase of International Application No. PCT/US2018/037452, filed on Jun. 14, 2018, which claims priority to U.S. Provisional Application No. 62/581,257, filed Nov. 3, 2017, and U.S. Provisional Application No. 62/520,011, filed Jun. 15, 2017, all of which are incorporated by reference herein in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Nov. 7, 2022 having the file name “18-613-WO-US-CON.xml” and is 538,000 bytes in size.

BACKGROUND

The high stability, diverse functionality, and favorable pharmacokinetic properties of macrocyclic peptides make them promising starting points for targeted therapeutics. However, there are few well-characterized natural macrocycles and they are difficult to repurpose for new functions. Thus most current approaches focus on random library selection methods, which, while powerful, only cover a small fraction of the vast sequence space that can be accessed by even short sequences of L- and D-amino acids, and often yield peptides which are not structured in the absence of target. Methods are needed for designing ordered macrocycles with shapes precisely crafted to bind their targets and with functionalities common in medicinal chemistry, but absent in the natural 20 amino acids, positioned at critical interaction sites.

SUMMARY

In one aspect are provided macrocyclic polypeptides comprising or consisting of a polypeptide having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids. In one embodiment, the polypeptides have at least one proline residue. In another embodiment, the polypeptides are between 7 and 14 amino acid residues in length, or between 7 and 10 amino acid residues in length. In a further embodiment, each amino acid substitution occurs at a non-proline position. In one embodiment, the amino acid substitutions do not include any non-proline residues being substituted with proline. In another embodiment, each amino acid substitution maintains the chirality of the amino acid replaced. In a further embodiment, each amino acid substitution is an alpha amino acid. In one embodiment, the polypeptides have at least 2, 3, 4, 5, 6, 7, 8, or more D amino acid residues. In another embodiment, the polypeptides have no more than 1 or 2 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof. In a further embodiment, the polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof.

In a further aspect the disclosure provides polypeptide libraries, comprising two or more polypeptides according to any embodiment or combination of embodiments of the disclosure.

In another aspect the disclosure provides for use of the polypeptides or the polypeptide libraries of any embodiment or combination of embodiments of the disclosure as a scaffold for target-based drug design or to screen molecules of interest for binding to one or more of the polypeptides.

DESCRIPTION OF THE FIGURES

FIG. 1 . 7-8 residue macrocycle NMR structures are very close to design models. Columns A: Design model, B: amino acid sequence, torsion bin string, hydrogen bond pattern and building block composition, C: observed backbone-backbone, backbone-sidechain) and sidechain-sidechain NOEs, D: overlay of design model on MD refined NMR ensemble (the average backbone rmsd to the NMR ensemble is indicated) for the design indicated at the bottom of column B. E: Average decrease in the propensity to favor the designed state (P_(Near), see methods) over all mutations at each position. Darker gray indicates larger decreases; positions particularly sensitive to mutation are boxed and indicated by color in the design model in column a. F: representative energy funnels for mutations at key positions as compared to the design sequence. Row I, column G: experimental SLIM data. Distribution of peak width at half height for peptide libraries with all amino substitutions at positions 4 and 5; the position 4 library has a broader distribution consistent with the computed energy landscape in column F. Rows II, IV, V, column G: Representative energy landscapes for double substitutions of critical residues overlaid on the original design landscape. Row III, column G: overlay of design model on alternative structure NMR ensemble (turn flip at bottom right).

FIG. 2 . 9-14 residue macrocycle NMR structures are very close to design models. Rows I-III: 9 and 10 residue designs. Columns A-G are as in FIG. 3 rows II, IV, V. Row IV: Comparison of bicyclic design models and NMR structures.

DETAILED DESCRIPTION

All references cited are herein incorporated by reference in their entirety.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid residues in D-form are noted with a “D” preceding the amino acid residue abbreviation. Amino acid residues in L-form are noted with just the amino acid residue abbreviation, noting that Glycine is non-chiral.

All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

In one aspect, the disclosure provides non-naturally occurring macrocyclic polypeptide comprising or consisting of a polypeptide having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids.

As shown in the examples that follow, the inventors have enumerated the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near exhaustive backbone sampling followed by sequence design and energy landscape calculations, and have identified 237 designs (SEQ ID NOS:1-237) predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. The polypeptides of the disclosure are attractive starting points for developing new therapeutics. One approach to inhibitor design is scaffolding loops at binding interfaces in the PDB; such scaffolding can increase binding affinity by pre-organizing the loops in the binding-competent conformation, enable additional interactions with the target, and improve cell permeability and oral bioavailability. In addition, due to their high stability and mutability, the polypeptides can be used as starting points in a library-based approach to find binders for molecules of interest.

As used herein, a macrocyclic polypeptide means a cyclic peptide of 7 to 14 amino acids in length. In various embodiments, the polypeptide may be 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-14, 9-13, 9-12, 9-11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, 13-14, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids in length. The polypeptides of the disclosure are N-to-C cyclized.

As will be understood by those of skill in the art, the polypeptides may be linked to other moieties (linkers, dyes, purification tags, peptides, small molecules, nucleic acids, etc.) as deemed appropriate for an intended use.

As used herein, a mirror image is a polypeptide with the same primary amino acid sequence as the reference sequence, but wherein each residue that is an L amino acid in the reference sequence is a D amino acid in the mirror image polypeptide, and wherein each residue that is an D amino acid in the reference sequence is an L amino acid in the mirror image polypeptide. A polypeptide and its mirror image share similar chemical and physical properties. The only difference is the chirality of the molecule.

In one embodiment, the polypeptide has at least one proline residue. In other embodiments, the peptides have at least 2, 3, or 4 proline residues. In one embodiment, a polypeptide of 7 amino acids in length has 0, 1, or 2 proline residues. In another embodiment, a polypeptide of 8 amino acids in length has 0, 1, or 2 proline residues. In another embodiment, a polypeptide of 9 amino acids in length has 0, 1, 2, 3, or 4 proline residues. In a further embodiment, a polypeptide of 10 amino acids in length has 0, 1, 2, 3, or 4 proline residues. In one embodiment, a polypeptide of 12 amino acids in length has 3 proline residues. In another embodiment, a polypeptide of 14 amino acids in length has 3 proline residues.

In one embodiment, the polypeptide has at least 2 D amino acids. In various further embodiments, the polypeptide has at least 3, 4, 5, 6, 7, 8, or more D amino acids. In one embodiment, a polypeptide of 7 amino acids in length has 2, 3, 4, or 5 D amino acids. In another embodiment, a polypeptide of 8 amino acids in length has 2, 3, 4, 5, or 6 D amino acids. In a further embodiment, a polypeptide of 9 amino acids in length has 2, 3, 4, 5, 6, or 7 D amino acids. In another embodiment, a polypeptide of 10 amino acids in length has 2, 3, 4, 5, 6, 7, or 8 D amino acids. In one embodiment, a polypeptide of 11 amino acids in length has 2, 3, 4, 5, 6, 7, 8, or 9 D amino acids. In another embodiment, a polypeptide of 12 amino acids in length has 2, 3, 4, 5, 6, 7, 8, 9, or 10 D amino acids. In one embodiment, a polypeptide of 13 amino acids in length has 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 D amino acids. In another embodiment, a polypeptide of 14 amino acids in length has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 D amino acids.

In another embodiment, the polypeptides have no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof. As shown in the examples that follow, the polypeptides of the disclosure are very amenable to mutation while maintaining structural stability and are highly protease stable. The amino acid substitutions may be any natural or unnatural amino acid. In other embodiments, the polypeptides have no more than 2 or 1 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof; in another embodiment, the polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof.

The amino acid substitutions may be any natural or unnatural amino acid. In one embodiment each amino acid substitution occurs at a non-proline position relative to the reference polypeptide. In a further embodiment, the amino acid substitutions do not include any non-proline residues being substituted to proline. In another embodiment, each amino acid substitution maintains the chirality of the amino acid replaced (i.e.: a D amino acid is replaced by a D amino acid or an L amino acid is replaced with an L amino acid). In a further embodiment, each amino acid substitution is an alpha amino acid (i.e.: amino acids having both the amine and the carboxylic acid groups attached to the first (alpha) carbon atom, which can be in L or D form, or glycine); this group includes all of the naturally occurring amino acids, selenocysteine, and pyrrolysine, and any unnatural amino acid that shares this backbone configuration.

Macrocyclic Polypeptides Designed as Described in the Examples: D Amino Acids Include a “D” Prior to the 3 Letter Amino Acid Abbreviation; L Amino Acids Just Show the 3 Letter Amino Acid Abbreviation

7-mers c.10.2.pdb (SEQ ID NO: 1) DHIS-PRO-DASP-DGLN-DSER-DGLU-PRO c.11.18.pdb (SEQ ID NO: 2) DARG-DLYS-PRO-DPRO-ASP-GLU-ASP c.2.8.pdb (SEQ ID NO: 3) PRO-ASN-DSER-DGLU-PRO-ASN-DASN c.3.100.pdb (SEQ ID NO: 4) DTHR-LYS-DASN-DASP-DTHR-ASN-PRO c.3.45.pdb (SEQ ID NO: 5) GLU-ASP-PRO-ARG-DLYS-TYR-DPRO c.4.35.pdb (SEQ ID NO: 6) DASP-ARG-GLN-PRO-DPRO-DASP-ASN c.4.59.pdb (SEQ ID NO: 7) DASP-DGLN-ASN-DGLU-DASN-PRO-PRO c.4.78.pdb (SEQ ID NO: 8) PRO-DASN-DTHR-ASN-DGLU-DASN-PRO c.5.4.pdb (SEQ ID NO: 9) GLN-ALA-PRO-ASP-DASN-ASN-DASP c.8.1.pdb (SEQ ID NO: 10) ASN-DLYS-DARG-PRO-DTHR-DASP-LYS c.9.2.pdb (SEQ ID NO: 11) ASP-DGLN-DASP-ARG-ARG-PRO-DPRO 8mer c.12.43.pdb (SEQ ID NO: 12) LYS-DTYR-DPRO-ASN-ASP-DGLN-DPRO-ASN c.15.21.pdb (SEQ ID NO: 13) DARG-GLU-DPRO-DGLN-ARG-DGLU-PRO-GLN c.16.34.pdb (SEQ ID NO: 14) PRO-ARG-ALA-DGLN-DTYR-PRO-ASP-DGLU c.16.48.pdb (SEQ ID NO: 15) PRO-ARG-ALA-DVAL-DHIS-GLU-ASP-DPRO c.17.51.pdb (SEQ ID NO: 16) DASP-GLU-DPRO-DGLN-GLU-DPRO-DASN-ASN c.18.79.pdb (SEQ ID NO: 17) PRO-DSER-DGLN-PRO-ARG-HIS-DLYS-DHIS c.2.47.pdb (SEQ ID NO: 18) DASP-DASN-DPRO-ASP-ASN-DASP-LYS-ASN c.21.27.pdb (SEQ ID NO: 19) TYR-DASP-GLN-DLEU-DPRO-PRO-LEU-DLYS c.24.83.pdb (SEQ ID NO: 20) ASP-GLU-PRO-ASN-DGLN-LYS-ASP-DASN c.28.17.pdb (SEQ ID NO: 21) DASN-ASP-ALA-PRO-DPRO-DALA-LYS-HIS c.28.65.pdb (SEQ ID NO: 22) DARG-DASP-GLU-ASP-PRO-ARG-DARG-ASP c.29.21.pdb (SEQ ID NO: 23) GLU-DTYR-PRO-DSER-DPRO-DTHR-DSER-DASN  c.29.5.pdb (SEQ ID NO: 24) DASN-ASN-ASP-DGLU-DPRO-DHIS-ARG-LYS c.4.31.pdb (SEQ ID NO: 25) PRO-LYS-DTHR-DGLU-PRO-ALA-DTHR-DASN c.43.15.pdb (SEQ ID NO: 26) DGLN-GLU-DALA-PRO-GLN-DASP-DPRO-DASN c.43.64.pdb (SEQ ID NO: 27) DLYS-LYS-DTHR-DGLU-PRO-DGLU-DGLU-DPRO c.44.97.pdb (SEQ ID NO: 28) DTHR-ASN-DASP-GLU-ALA-PRO-DSER-DPRO c.45.36.pdb (SEQ ID NO: 29) DGLU-PRO-DALA-DLYS-ASP-DLYS-DHIS-LYS c.5.40.pdb (SEQ ID NO: 30) DLYS-DVAL-PRO-DASP-DGLN-DILE-PRO-DASN c.64.23.pdb (SEQ ID NO: 31) DSER-LYS-GLU-LYS-DTHR-ASP-DPRO-GLU  tlc.164.98.pdb (SEQ ID NO: 32) ASP-ASP-PRO-THR-DPRO-DARG-GLN-DGLN; also referred to as design 8.1 9mer t6c.105.6.pdb (SEQ ID NO: 33) ASP-ASN-LYS-DASP-HIS-DPRO-ASN-ASP-DLYS t6c. 109.55.pdb (SEQ ID NO: 34) DHIS-LYS-DSER-DPRO-DSER-DLYS-SER-DGLU-ASP t6c.11.47.pdb (SEQ ID NO: 35) DILE-DPRO-PRO-DVAL-ILE-GLU-DASN-DASP-GLN t6c.11.93.pdb (SEQ ID NO: 36) PRO-DARG-LYS-DLEU-DPRO-ASP-GLU-DGLN-DSER t6c.111.45.pdb (SEQ ID NO: 37) PRO-DSER-DASN-GLU-DARG-ASP-ASP-DTHR-GLN t6c. 112.7.pdb (SEQ ID NO: 38) GLN-PHE-PRO-DASP-THR-DLYS-ASP-DALA-DASP t6c. 112.74.pdb (SEQ ID NO: 39) DARG-DALA-DPRO-PRO-LYS-PRO-DASP-LYS-DASP t6c.116.43.pdb (SEQ ID NO: 40) VAL-DGLN-PRO-DPRO-ALA-DTHR-ASP-GLU-SER t6c. 125.31.pdb (SEQ ID NO: 41) DPRO-ALA-DGLU-PRO-ASN-DTHR-DLYS-SER-PRO t6c. 129.81.pdb (SEQ ID NO: 42) DGLN-GLN-PRO-DILE-DPRO-ASP-DALA-ASP-ASP t6c. 136.68.pdb (SEQ ID NO: 43) GLN-HIS-PRO-DGLU-PRO-PRO-SER-LEU-DASP t6c. 14.12.pdb (SEQ ID NO: 44) HIS-ALA-DGLN-ASP-ASN-DASP-DPRO-DSER-DLYS t6c. 14.24.pdb (SEQ ID NO: 45) DASP-DASN-LYS-SER-DGLN-ASP-ASN-DVAL-DASP t6c. 154.74.pdb (SEQ ID NO: 46) DPRO-THR-DTHR-GLU-LYS-ASP-DVAL-PRO-DGLN t6c.168.7.pdb (SEQ ID NO: 47) PRO-DASN-DASP-ALA-PRO-DPRO-GLU-PRO-LEU t6c. 171.34.pdb (SEQ ID NO: 48) PRO-PRO-DTHR-ALA-PRO-DPRO-DASP-ASP-DLYS t6c.18.44.pdb (SEQ ID NO: 49) DGLU-DASN-PRO-DPRO-DILE-DALA-DPRO-ASP-ASN t6c. 183.50.pdb (SEQ ID NO: 50) DPRO-DASN-ASP-DSER-DASP-LYS-PRO-DASN-DSER t6c. 187.12.pdb (SEQ ID NO: 51) DVAL-DASP-ASP-DASP-HIS-PRO-DARG-DPRO-ASN t6c.22.15.pdb (SEQ ID NO: 52) DASP-LYS-DTHR-DASN-ASP-PRO-DPRO-ALA-LYS t6c.23.91.pdb (SEQ ID NO: 53) PRO-DPRO-SER-DSER-DSER-DASN-LYS-DSER-DARG t6c.238.6.pdb (SEQ ID NO: 54) DPRO-ASN-TYR-DHIS-PRO-LYS-ASP-LEU-DGLN t6c.244.59.pdb (SEQ ID NO: 55) DTHR-GLN-DASN-ASN-DASP-DPRO-DARG-DSER-SER t6c.26.74.pdb (SEQ ID NO: 56) PRO-DASN-DASP-GLN-DPRO-ASN-DLYS-GLU-HIS t6c.26.78.pdb (SEQ ID NO: 57) DPRO-PRO-DASP-ASP-DASP-LYS-PRO-DASN-LYS t6c.31.88.pdb (SEQ ID NO: 58) DPRO-LYS-ASP-DTHR-DASP-GLN-GLU-DPRO-GLU t6c.32.76.pdb (SEQ ID NO: 59) PRO-DPRO-DTYR-DPRO-ASP-SER-ARG-DILE-ALA t6c.32.9.pdb (SEQ ID NO: 60) DVAL-LEU-ASP-ASP-SER-DVAL-VAL-DPRO-PRO t6c.33.60.pdb (SEQ ID NO: 61) PRO-DGLU-SER-DALA-LYS-DASP-ASP-DLEU-DASN t6c.33.8.pdb (SEQ ID NO: 62) DPRO-GLU-DTHR-DLYS-DPRO-ASN-VAL-DVAL-PRO t6c.38.39.pdb (SEQ ID NO: 63) DALA-DLYS-HIS-DASN-HIS-ASP-DLYS-ASP-ASN t6c.40.21.pdb (SEQ ID NO: 64) LYS-DGLN-DASP-PRO-DARG-HIS-ASP-DLYS-ASP t6c.40.92.pdb (SEQ ID NO: 65) DSER-TYR-DGLN-ASP-ASN-DALA-DILE-ASN-DTHR t6c.54.36.pdb (SEQ ID NO: 66) DGLN-DPRO-ASN-VAL-DASP-LYS-DASP-DASN-THR t6c.54.87.pdb (SEQ ID NO: 67) ASP-DVAL-PRO-DPRO-ALA-DGLU-ARG-PRO-DPRO t6c.54.93.pdb (SEQ ID NO: 68) DPRO-DASP-ASN-DVAL-PRO-DPRO-THR-DVAL-DLYS t6c.58.11.pdb (SEQ ID NO: 69) VAL-DARG-PRO-DSER-VAL-DGLN-DGLU-DPRO-DASN t6c.6.97.pdb (SEQ ID NO: 70) ALA-PRO-DSER-DALA-ASP-DGLN-DASN-DPRO-ASN t6c.61.79.pdb (SEQ ID NO: 71) VAL-PRO-ASP-DARG-DVAL-LEU-PRO-DASN-DTYR t6c.62.76.pdb (SEQ ID NO: 72) DTHR-DASP-DGLN-ASP-GLU-PRO-DTHR-LYS-GLU t6c.76.60.pdb (SEQ ID NO: 73) ASP-PRO-ASN-DLYS-ASP-ASP-ARG-DTHR-DTYR t6c.8.39.pdb (SEQ ID NO: 74) DPRO-ASP-ASN-DSER-PRO-THR-GLN-DGLN-DTYR t6c.80.74.pdb (SEQ ID NO: 75) DSER-DPRO-DSER-ASP-DGLN-ASP-SER-SER-SER t6c.81.48.pdb (SEQ ID NO: 76) DILE-DPRO-ASP-DARG-THR-DASP-DASP-SER-LYS t6c.83.33.pdb (SEQ ID NO: 77) DPRO-ASN-GLN-DASN-GLN-DASP-DLEU-DPRO-DILE t6c.85.94.pdb (SEQ ID NO: 78) ASP-DGLU-DPRO-ASN-DGLN-PRO-DASN-ASP-DASP t6c.9.57.pdb (SEQ ID NO: 79) PRO-DTHR-DASP-ASP-GLU-DASN-THR-DLYS-HIS t6c.9.91.pdb  (SEQ ID NO: 80) GLU-DLYS-ASN-SER-ASN-DGLU-LYS-PRO-DPRO t6c.96.89.pdb  (SEQ ID NO: 81) DPRO-DASP-GLN-TYR-DARG-ASP-DPRO-TYR-DASP 10mer  c.100.22.pdb  (SEQ ID NO: 82) DASP-DASP-DGLU-LYS-DLYS-ASN-DGLU-PRO-ASP-DALA c.100.72.pdb  (SEQ ID NO: 83)  DGLN-DGLU-DASP-ARG-DTHR-DGLU-DGLU-PRO-ARG-DARG c. 1003.22.pdb  (SEQ ID NO: 84) DTYR-PRO-ALA-DGLN-DPRO-PRO-DLEU-LEU-DLYS-ASP c.102.56.pdb (SEQ ID NO: 85) DASN-DLYS-DGLU-DLYS-DASP-LYS-ALA-PRO-DGLU-PRO c.1032.1.pdb (SEQ ID NO: 86) DGLU-DPRO-ASP-DLYS-PRO-DASN-ALA-ASP-DGLN-DGLN c.105.97.pdb (SEQ ID NO: 88) ASN-DSER-DLYS-DASP-ASP-DTHR-DGLU-PRO-DASN-DPRO c.1056.21.pdb (SEQ ID NO: 89) DPRO-GLU-PRO-DGLU-DPRO-DVAL-PRO-ALA-DLYS-DALA c.106.6.pdb (SEQ ID NO: 90) DPRO-DARG-DALA-LYS-LEU-PRO-DASN-DSER-DASP-ALA c.107.22.pdb (SEQ ID NO: 91) GLU-DPRO-PRO-ASN-ALA-LYS-ASP-DASN-ASN-ALA c.107.77.pdb (SEQ ID NO: 92) LYS-DASP-GLN-DPRO-PRO-GLN-ARG-LYS-ASP-DASN c. 1078.20.pdb (SEQ ID NO: 93) DARG-DASP-LYS-ASP-DLYS-DGLU-PRO-DPRO-ASP-DALA c.109.44.pdb (SEQ ID NO: 94) DGLU-DASN-PRO-ALA-DLYS-LYS-PRO-DASP-DHIS-LYS c.1095.10.pdb (SEQ ID NO: 95) LYS-ASN-DPRO-PRO-PRO-DTHR-DGLU-PRO-ALA-ALA c.110.32.pdb (SEQ ID NO: 96) DALA-DPRO-ASN-TYR-DSER-DLYS-ASP-ASN-DPRO-DLYS c.110.61.pdb (SEQ ID NO: 97) LEU-PRO-ARG-DGLN-DPRO-ASN-ASP-DSER-DLYS-DTHR c.110.87.pdb (SEQ ID NO: 98)  GLU-DPRO-DASN-DSER-DGLU-DPRO-ASN-DASP-DSER-DASN c.111.100.pdb (SEQ ID NO: 99) DLYS-DASP-DASN-ASP-PRO-ASN-ASN-DLYS-DLEU-ASP c.111.82.pdb (SEQ ID NO: 100) PRO-DASN-GLU-PRO-LYS-TYR-DLYS-DASN-ASP-DGLU c.112.45.pdb (SEQ ID NO: 101) ALA-LYS-ASP-DLYS-ASP-ASN-LYS-DASP-PRO-LYS c.112.88.pdb (SEQ ID NO: 102) GLN-GLN-DASP-DASP-LYS-ASP-GLN-PRO-DPRO-ASP c.113.66.pdb (SEQ ID NO: 103) DGLU-DGLU-PRO-LYS-DILE-PRO-ASP-DLYS-DGLU-DILE c.114.4.pdb (SEQ ID NO: 104) DPRO-ASP-DVAL-LYS-PRO-DPRO-GLU-DLEU-LYS-PRO c.1143.27.pdb (SEQ ID NO: 105) GLU-GLU-DSER-DPRO-DSER-DSER-DPRO-ASN-DTHR-ASP c.115.8.pdb (SEQ ID NO: 106) LYS-ASP-DGLN-DPRO-LYS-DASN-PRO-DASP-DGLN-PHE c.1178.14.pdb (SEQ ID NO: 107) ARG-TYR-DSER-TRP-DARG-DASP-PRO-TYR-DGLN-PRO c.1181.8.pdb (SEQ ID NO: 108) DTYR-ASP-PRO-ARG-DASP-DSER-DLYS-GLN-DPRO-ASN c.1187.26.pdb (SEQ ID NO: 109) ASN-DTYR-DPRO-ASP-PRO-ARG-DTYR-DPRO-ASP-PRO c.119.73.pdb (SEQ ID NO: 110) GLN-ARG-ASN-HIS-DPRO-ASP-DTHR-GLN-DPRO-ASP c.12.37.pdb (SEQ ID NO: 111) DLEU-GLN-DTHR-DARG-PRO-DSER-ALA-GLU-PRO-DASP c.120.11.pdb (SEQ ID NO: 112) GLN-DTYR-LYS-HIS-DASP-HIS-PRO-DHIS-PRO-DASP c.120.33.pdb (SEQ ID NO: 113) ALA-ASN-DASN-HIS-PRO-DASN-ALA-DASP-PRO-DALA c.128.3.pdb (SEQ ID NO: 115) DLYS-ASP-ASN-DPRO-ASN-DALA-DASP-PRO-DLYS-ASP c.129.40.pdb (SEQ ID NO: 116) PRO-DARG-DASP-GLN-GLU-DPRO-ASN-DSER-DSER-DASN c.1299.4.pdb (SEQ ID NO: 117) LEU-DVAL-ARG-DASN-HIS-PRO-DPRO-ASP-DGLU-ASN c.137.2.pdb (SEQ ID NO: 118) DGLN-DALA-PRO-ASN-LYS-DARG-LYS-DPRO-ASP-ASP c.138.17.pdb (SEQ ID NO: 119) ALA-PRO-DSER-DILE-GLN-PRO-ASN-DGLU-DASN-ASN c.140.60.pdb (SEQ ID NO: 120) ASN-ASN-DLYS-ASP-ASN-DASP-PRO-ALA-DARG-PRO c.142.41.pdb (SEQ ID NO: 121)  PRO-DPRO-GLU-DALA-DARG-GLU-GLU-DPRO-DALA-DGLN c.143.37.pdb (SEQ ID NO: 122) DTYR-DPRO-HIS-PRO-DASN-DTYR-GLU-ASP-LYS-ASP c.143.85.pdb (SEQ ID NO: 123) DGLN-DPRO-ASP-PRO-ASN-DVAL-GLU-MET-LYS-ASP c.145.1.pdb (SEQ ID NO: 124) ASP-PRO-DASN-DLYS-LYS-GLU-DASP-GLU-ASN-DSER c.145.61.pdb (SEQ ID NO: 125) DASN-DALA-GLN-ASP-DASN-PRO-DGLU-DPRO-LYS-PRO c.146.71.pdb (SEQ ID NO: 126) DPRO-ASP-GLN-DASP-ASP-PRO-ARG-ARG-DSER-DALA c.148.21.pdb (SEQ ID NO: 127) DHIS-ASN-DSER-GLU-ALA-DASN-PRO-ASN-ARG-DALA c.148.33.pdb (SEQ ID NO: 128) DASN-ASP-DGLN-DLYS-DASP-ASN-DSER-DGLU-PRO-PRO c.148.90.pdb (SEQ ID NO: 129) GLU-TYR-DPRO-DLYS-DSER-ALA-ALA-PRO-LYS-DGLN c.15.52.pdb (SEQ ID NO: 130) PRO-DHIS-DPRO-ASN-ASP-DVAL-ASN-ASN-DASN-ARG c.151.53.pdb (SEQ ID NO: 131) DTYR-DPRO-ASP-TYR-DILE-DPRO-ASP-ASP-ARG-TYR c.153.54.pdb (SEQ ID NO: 132) DSER-LYS-DASP-ALA-PRO-GLU-DGLU-PRO-ARG-ARG c.154.1.pdb (SEQ ID NO: 133) LYS-DGLU-PRO-DSER-DSER-DALA-DGLU-PRO-ASN-DASP c.155.55.pdb (SEQ ID NO: 134)  DSER-DPRO-DALA-LYS-DPRO-DASN-DSER-GLN-PRO-DASN c.157.24.pdb (SEQ ID NO: 135) ASP-ASN-LYS-DASN-PRO-DPRO-DASP-DGLN-DSER-DGLN c.157.39.pdb (SEQ ID NO: 136) ASP-DSER-PRO-ASN-LEU-DSER-DASP-GLN-DGLN-DPRO c.157.61.pdb (SEQ ID NO: 137) ASP-DSER-PRO-ASN-LEU-ASN-LYS-ASP-DVAL-DPRO c.157.63.pdb (SEQ ID NO: 138) DTHR-DGLU-PRO-DGLN-DSER-GLU-DPRO-PRO-ASN-LEU c.158.36.pdb (SEQ ID NO: 139)  ASP-DGLU-DALA-DPRO-ASN-LYS-DGLU-DARG-DPRO-ASN c.159.6.pdb (SEQ ID NO: 140) ASN-DLYS-LEU-PRO-PRO-DASP-ALA-DTHR-DASN-DGLU c.16.12.pdb (SEQ ID NO: 141) DARG-DLYS-DGLU-PRO-DALA-GLU-ASP-DASN-PRO-ASN c.16.3.pdb (SEQ ID NO: 142) PRO-ASN-DARG-DTHR-DGLU-PRO-DALA-GLU-TYR-DASP c.16.31.pdb (SEQ ID NO: 143) DLEU-DPRO-GLU-DPRO-DTYR-ALA-LEU-DLYS-PRO-ASN c.161.54.pdb (SEQ ID NO: 144) LYS-DSER-PRO-DPRO-DASN-ASP-ASN-LYS-ASP-DVAL c.161.55.pdb (SEQ ID NO: 145) DVAL-PRO-ASP-HIS-ASN-DASN-PRO-ASP-HIS-ASN c. 164.11.pdb (SEQ ID NO: 146)  LYS-GLU-DVAL-DPRO-ASN-DTHR-DSER-DPRO-DSER-DALA c.164.35.pdb (SEQ ID NO: 147)  DTHR-DASP-DASP-ASP-DGLN-ALA-DILE-DPRO-PRO-DVAL c.165.18.pdb (SEQ ID NO: 148)  DLYS-DARG-LYS-DLEU-DPRO-GLU-PRO-DGLU-GLU-DALA c.165.81.pdb (SEQ ID NO: 149)  GLU-DPRO-ASP-DSER-DSER-DASN-GLU-DTYR-PRO-DARG c.17.74.pdb (SEQ ID NO: 150) ASP-LYS-DLYS-DLEU-ALA-PRO-DASN-DASP-ASP-PRO c.175.67.pdb (SEQ ID NO: 151) DPRO-DALA-DSER-ASP-PRO-ARG-DARG-GLU-DGLN-PRO c.177.32.pdb (SEQ ID NO: 152) GLU-DALA-DLYS-ASP-DVAL-DPRO-ASP-ASN-MET-DPRO c.180.41.pdb (SEQ ID NO: 153) MET-ASN-LYS-DLYS-PRO-DASP-ALA-DTHR-PRO-ASP c.185.87.pdb (SEQ ID NO: 154) ALA-DGLN-TYR-PRO-DASP-GLN-DARG-DGLN-PRO-ALA c.186.82.pdb (SEQ ID NO: 155) PRO-DHIS-LYS-GLN-PRO-DASP-DASP-ASN-DASN-GLU c.187.91.pdb (SEQ ID NO: 156) DASP-ALA-PRO-DPRO-ASN-ASP-DASP-DASN-PRO-DSER c.19.76.pdb (SEQ ID NO: 157) DLYS-ASN-DASN-DASP-GLN-DASP-DLYS-TYR-PRO-DPRO c.191.37.pdb (SEQ ID NO: 158)  ASN-DVAL-ASN-PRO-DTYR-DPRO-ASP-DALA-DPRO-DPRO c.195.98.pdb (SEQ ID NO: 159) GLN-DPRO-DPRO-ASN-DALA-PRO-LYS-GLU-DSER-DSER c.2.21.pdb (SEQ ID NO: 160) ASN-DALA-PRO-ASN-DTHR-DSER-DASP-GLU-ASN-DLYS c.20.98.pdb (SEQ ID NO: 161)  GLN-DGLU-PRO-DPRO-ALA-DALA-DALA-GLN-DASP-DLYS c.200.97.pdb (SEQ ID NO: 162) ASP-DSER-DPRO-DSER-DASN-ASP-PRO-ARG-HIS-DASP c.201.15.pdb (SEQ ID NO: 163) DVAL-ASP-HIS-LYS-DGLN-PRO-DPRO-ALA-DLYS-GLU c.205.19.pdb (SEQ ID NO: 164) DSER-DPRO-DSER-DLYS-ASP-DLYS-DASP-ASN-ALA-PRO c.206.81.pdb (SEQ ID NO: 165) DARG-DPRO-ASP-DASP-PRO-ASN-DASP-DLYS-DARG-ASP c.206.85.pdb (SEQ ID NO: 166) ALA-LEU-DGLU-PRO-ASN-DSER-DPRO-DSER-GLU-DSER c.212.79.pdb (SEQ ID NO: 167) DSER-ASP-DGLN-TYR-DPRO-ASN-DALA-DPRO-ASP-ASP c.213.68.pdb (SEQ ID NO: 168) DGLU-DALA-ARG-ASP-HIS-LYS-DVAL-PRO-DPRO-ALA c.217.29.pdb (SEQ ID NO: 169) GLN-ASP-ASN-DLYS-ASP-DGLN-ASP-ASN-PRO-ASP c.217.71.pdb (SEQ ID NO: 170) TYR-PRO-GLU-ALA-LYS-ASP-DASN-ASN-LYS-DASP c.22.67.pdb (SEQ ID NO: 171) PRO-ASP-DTHR-DARG-DASP-ALA-DGLN-ASP-ARG-DILE c.223.66.pdb (SEQ ID NO: 172) LYS-PRO-GLN-GLU-DPRO-PRO-DASP-ALA-ASN-LYS c.224.14.pdb (SEQ ID NO: 173) ASP-DVAL-ASP-PRO-DGLU-DHIS-DPRO-ASN-DALA-DLYS c.225.68.pdb (SEQ ID NO: 174)  GLU-DPRO-ASN-DASP-DPRO-ASN-DASN-DGLU-PRO-DVAL c.229.8.pdb (SEQ ID NO: 175) DPRO-ASN-DASP-DGLU-PRO-ASP-LYS-DASP-ARG-DHIS c.231.18.pdb (SEQ ID NO: 176)  DSER-GLU-DPRO-DGLN-GLN-DSER-GLU-DPRO-DALA-TYR c.234.57.pdb (SEQ ID NO: 177) DPRO-ALA-DASP-HIS-LYS-ASN-DARG-LYS-DGLU-PRO c.24.60.pdb (SEQ ID NO: 178) DASP-ASP-DGLN-LEU-PRO-DASP-DVAL-PRO-ASN-ALA c.24.90.pdb (SEQ ID NO: 179) DARG-DSER-PRO-GLU-LYS-DSER-DLYS-ASP-LYS-PRO c.241.1.pdb (SEQ ID NO: 180) PRO-ASN-LYS-DASP-ASN-DGLU-PRO-ALA-ARG-DGLU c.241.69.pdb (SEQ ID NO: 181) PRO-ASN-LYS-DASP-GLN-PRO-DSER-ALA-ASP-DGLU c.241.95.pdb (SEQ ID NO: 182) ALA-ASP-DARG-TYR-DASP-DGLU-PRO-MET-PRO-DSER c.244.45.pdb (SEQ ID NO: 183) LYS-ASN-DLYS-DSER-DGLU-PRO-PRO-DASP-PRO-ALA c.244.98.pdb (SEQ ID NO: 184) ASP-GLU-ARG-PRO-DPRO-LYS-ALA-LYS-ASP-DLYS c.257.63.pdb (SEQ ID NO: 185) DALA-ASP-DARG-DASN-ASP-PRO-ARG-ALA-DTHR-DSER c.257.93.pdb (SEQ ID NO: 186) GLN-ALA-PRO-DGLU-PRO-PRO-GLU-ALA-DLYS-DASP c.264.71.pdb (SEQ ID NO: 187)  ASN-DTYR-DGLU-DPRO-HIS-DLYS-DTYR-ASP-DLEU-DPRO c.265.16.pdb (SEQ ID NO: 188) DTHR-PRO-LYS-DTHR-ASP-DLYS-ASP-ARG-DASP-DPRO c.268.11.pdb (SEQ ID NO: 189)  ALA-DASP-DPRO-DSER-LYS-DGLU-DLEU-DPRO-ASP-DASN c.27.32.pdb (SEQ ID NO: 190) DGLU-PRO-DPRO-ALA-LYS-ASP-DHIS-ASN-DASP-ARG c.28.81.pdb (SEQ ID NO: 191) ASP-ALA-PRO-LYS-PRO-DSER-DGLN-GLN-DASP-DASN c.285.52.pdb (SEQ ID NO: 192)  DGLN-ASN-DGLU-ASN-DALA-HIS-GLN-DASP-DPRO-DARG c.287.72.pdb (SEQ ID NO: 193) ASN-LYS-DGLN-PRO-DASP-ASN-DTHR-ASN-ASP-DPRO c.29.27.pdb (SEQ ID NO: 194)  DPRO-DASN-DALA-ASN-GLN-DARG-DPRO-PRO-DASP-GLN c.29.82.pdb (SEQ ID NO: 195)  ASN-DTYR-ASN-DGLU-DASN-DALA-GLN-HIS-DPRO-DPRO c.292.61.pdb (SEQ ID NO: 196)  DPRO-DVAL-LYS-DASP-DASP-DHIS-PRO-DASN-DASP-GLU c.292.81.pdb (SEQ ID NO: 197) DGLN-DASN-DPRO-ASN-ASN-PRO-ARG-DLYS-DALA-ASP c.294.7.pdb (SEQ ID NO: 198)  ASP-DLYS-ASP-DTYR-DGLU-PRO-DPRO-DTHR-DALA-DHIS c.3.70.pdb (SEQ ID NO: 199) ASP-DASN-ALA-PRO-ASN-DASP-LYS-ASP-DGLN-DSER c.306.55.pdb (SEQ ID NO: 200) TYR-GLU-DTYR-PRO-DASP-DLEU-DPRO-DILE-PRO-DSER c.306.8.pdb (SEQ ID NO: 201) DPRO-PRO-PRO-GLU-ASN-DSER-DLEU-ASP-DGLN-DLEU c.31.79.pdb (SEQ ID NO: 202) DASN-GLU-ALA-GLU-PRO-LYS-DSER-DALA-ALA-ASP c.310.87.pdb (SEQ ID NO: 203) GLU-PRO-LYS-TYR-DASP-GLN-ASP-MET-ARG-ARG c.312.72.pdb (SEQ ID NO: 204) ASP-ASP-PRO-ARG-LYS-DASP-ASP-ALA-GLN-DASP c.315.84.pdb (SEQ ID NO: 205) DGLU-DTHR-LYS-DALA-DPRO-DTHR-DGLU-DGLU-PRO-DLYS c.326.62.pdb (SEQ ID NO: 206) DGLN-ALA-DARG-GLN-PRO-DPRO-ASP-ALA-ASN-LYS c.33.10.pdb (SEQ ID NO: 207) DGLU-PRO-ASN-DVAL-DASN-DGLU-DPRO-DARG-LYS-ALA c.33.75.pdb (SEQ ID NO: 208) DSER-DGLU-DPRO-DASP-DASN-LYS-ALA-DLYS-PRO-ASN c.33.80.pdb (SEQ ID NO: 209) DALA-LYS-GLU-DGLN-ASP-ALA-DGLN-ALA-PRO-DPRO c.334.4.pdb (SEQ ID NO: 210) PRO-ASN-DLYS-ASP-DSER-DPRO-DLYS-LYS-ASP-DVAL c.339.46.pdb (SEQ ID NO: 211) DSER-DASP-DSER-DGLN-LYS-PRO-DPRO-LYS-DLEU-ASP c.339.9.pdb (SEQ ID NO: 212) DTYR-PRO-TYR-PRO-DASP-DHIS-ALA-ASP-DGLN-LYS c.34.5.pdb (SEQ ID NO: 213) DVAL-DPRO-ASN-DTRP-GLU-DPRO-TYR-DGLN-ASP-LYS c.34.99.pdb (SEQ ID NO: 214) DLYS-ASP-DALA-DPRO-PRO-ALA-DLYS-ASP-ARG-DASN c.340.84.pdb (SEQ ID NO: 215) GLN-ASP-DLYS-GLU-DALA-DPRO-PRO-LYS-ASP-DASP c.341.76.pdb (SEQ ID NO: 216) DALA-GLN-DGLU-PRO-ALA-DGLN-ASP-HIS-PRO-DASN c.342.15.pdb (SEQ ID NO: 217)  GLN-DPRO-DARG-DALA-LYS-ALA-DLYS-DGLU-DPRO-DLYS c.344.36.pdb (SEQ ID NO: 218) DASP-DASP-ARG-DLYS-PRO-DGLU-PRO-DLYS-DPRO-ASP c.346.38.pdb (SEQ ID NO: 219) ASP-DASP-GLN-PRO-DASP-ASP-DASP-GLN-PRO-DASP c.351.67.pdb (SEQ ID NO: 220) DPRO-ASN-DILE-DASP-DPRO-ASP-PRO-ARG-DASN-DARG c.352.6.pdb (SEQ ID NO: 221) DGLN-ASP-LYS-GLU-DPRO-DASP-PRO-ASN-ALA-ASP c.356.41.pdb (SEQ ID NO: 222) ASP-DGLU-PRO-ASN-ALA-GLU-DSER-DPRO-DSER-GLN c.358.11.pdb (SEQ ID NO: 223) DLYS-DGLU-DLYS-ASP-DLYS-DPRO-ASP-PRO-ARG-GLN c.362.67.pdb (SEQ ID NO: 224) DPRO-ASN-ASP-ALA-PRO-DASP-LYS-DASP-DASN-DGLN c.369.88.pdb (SEQ ID NO: 225) DGLN-PRO-DASN-DALA-DPRO-LYS-DTHR-GLU-TRP-ALA 11_SS (SEQ ID NO: 226) GLU-DPRO-PRO-ALA-LYS-ASP-ASN-DLYS-DSER-DSER c.38.19.pdb (SEQ ID NO: 227)  DALA-GLN-DPRO-DCYS-DLYS-ASP-SER-DTYR-DCYS-PRO-DSER 12_SS (SEQ ID NO: 228)  HIS-DPRO-DVAL-CYS-DLEU-PRO-DPRO-GLU- DLYS-VAL-CYS-DGLU 14_SS (SEQ ID NO: 229) DPRO-DCYS-ASN-DVAL-DPRO-ASP-VAL-TYR-CYS- DPRO-DASN-LYS-TYR-DVAL 7.1 (SEQ ID NO: 230) ASP-THR-DASN-DPRO-THR-DLYS-ASN 7.2 (SEQ ID NO: 231) ASP-GLN-SER-GLU-DPRO-HIS-DPRO 7.3 (SEQ ID NO: 232) GLN-ASP-PRO-DPRO-LYS-DTHR-DASP 7.4 (SEQ ID NO: 233) DLYS-TYR-DPRO-GLU-ASP-GLU-ARG 8.2 (SEQ ID NO: 234) PRO-GLN-DARG-GLN-DPRO-DGLN-ARG-DGLU 9.1 (SEQ ID NO: 235) LYS-ASP-LEU-DGLN-DPRO-PRO-TYR-DHIS-PRO 10.1 (SEQ ID NO: 236) PRO-GLU-ALA-ALA-ARG-DVAL-DPRO-ARG-DLEU-DTHR 10.2 (SEQ ID NO: 237) GLU-DVAL-ASP-PRO-DGLU-DHIS-DPRO-ASN-DALA-DPRO

The polypeptides of the disclosure can be made by any suitable technique, including but not limited to the methods disclosed in the examples that follow.

In another embodiment, the disclosure provides polypeptide libraries, comprising two or more polypeptides according to any embodiment or combination of embodiments of the disclosure. The polypeptide libraries can be used for any suitable purpose, including but not limited to screening for suitable polypeptides to serve as scaffolds for therapeutic development. In various embodiments, the libraries comprise 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 237, 250, 275, 300, 325, 350, 375, 400, 425, 450, 474, 500, 750, 1000, or more of the polypeptides of the disclosure.

The polypeptide libraries may be present in solution or on a solid support including but not limited to a microarray, glass slide, membrane, microplate, beads, or resins. The polypeptides in the library may be labeled with a detectable label. The libraries may be stored frozen, in lyophilized form, or as a solution.

In another embodiment the disclosure provides uses of the polypeptides or polypeptide libraries of any embodiment or combination of embodiments of the disclosure as a scaffold for target-based drug design, or as a starting point in a library-based approach to find binders for molecules of interest.

Examples

Mixed chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to-date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: peptide macrocycles lack regular secondary structures and hydrophobic cores and can contain local structures not accessible with L-amino acids. Here we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. NMR structures of nine of twelve designed 7-10 residue macrocycles, and three 11-14 residue bicyclic designs are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.

The high stability, diverse functionality, and favorable pharmacokinetic properties of macrocyclic peptides make them promising starting points for targeted therapeutics (1-4). However, there are few well-characterized natural macrocycles and they are difficult to repurpose for new functions. Designing shorter peptide macrocycles had remained an unsolved challenge. The driving force for the folding of larger peptides and proteins is the sequestration of hydrophobic residues in a non-polar core, enabled by regular secondary structures in which buried backbone polar groups can make hydrogen bonds. This principle has been the basis of almost all previous peptide and protein design work. However, the balance of forces is considerably different for 7-14 residue peptides: they are too small to have either a solvent-excluded hydrophobic core or α-helical and β-sheet (other than (3-hairpin) secondary structures. Beyond these differences in the physics of folding, protein design methods often use the PDB (Protein Data Bank) as a source of local structural information, but native structures provide a poor guide for local structures that include non-canonical D-amino acids. On the other hand, short cyclic peptides are an attractive target for computational design as unlike larger systems, there is the possibility of obtaining a completeness of conformational sampling rare in any molecular design endeavor.

The local structure space relevant for cyclic peptides is quite different than that of proteins, so they cannot be systematically generated by assembling protein fragments. Instead, we used generalized kinematic closure (genKIC) methods (15-17) with achiral flat-bottom backbone torsional sampling distributions to generate closed backbone structures starting from a polyglycine chain. For each structure, we used Monte Carlo simulated annealing to search for the lowest energy amino acid sequence, restricting positions with negative values of the backbone torsion angle phi to L-amino acids (and rotamers) and those with positive values to D-amino acids, and disallowing glycine to maximize local sequence encoding of the structure. In preliminary calculations, we found that energy gaps greater than ˜10 k_(B)T (˜6 kcal/mol) could only be obtained for N-residue macrocycles if they contained at least N/3 backbone hydrogen bonds; hence in subsequent calculations backbones with fewer hydrogen bonds were discarded. We carried out large scale backbone generation and sequence design calculations for 7-10 residue backbone cyclized peptides, obtaining 50, 596, 12374, 49571 distinct backbones for lengths 7, 8, 9 and 10 respectively after clustering based on backbone torsion angle bins (ABXY, where torsion bin A=the helical region of Ramachandran space, B=extended strand-like region, X=mirror of A, Y=mirror of B) and backbone hydrogen bond patterns. Because the sampling method is stochastic, there is no guarantee of completeness, but the symmetry of the system enables a convergence test: for each distinct peptide backbone conformation identified, the mirror image should also be observed. As the amount of sampling increases, the number of clusters identified for which the mirror image is observed initially increases, as does the number of clusters with no mirror. The former then plateaus, while the latter decreases to near zero. Such convergence suggests near-complete coverage of the combined D- and L-space compatible with peptide closure with backbone hydrogen bonds and no steric clashes. We also sampled and designed structures for 11-14 residue macrocycles, but did not seek completeness due to combinatorial explosion in the number of states.

The Monte Carlo simulated annealing sequence design calculations seek a sequence that minimizes the energy of the target backbone conformation, but there is no guarantee that the sequence found maximizes the energy gap between the target backbone conformation and alternative conformations. To assess the energy landscape for low energy designs (from 21 designs for length 7 to 673 designs for length 10), 105-106 conformations were generated for each sequence, and the energy minimized with respect to the backbone and sidechain torsion angles. The energy gap and Boltzmann-weighted probability of finding the peptide in or close to the designed main chain conformation (P_(Near)) were estimated from the resulting energy landscapes. A total of 12, 22, 45, and 145 designs with distinct backbone structures had energy landscapes strongly funneled into the design target structure for 7, 8, 9 and 10 residue macrocycles respectively

Because of the constraints imposed by the cyclic backbone, the small size, and the presence of D-amino acids, the designs span a local structural space inaccessible or underexplored in native proteins. Recurrent features include hydrogen-bonded turn-like structures and proline-stabilized kinks, some of which are observed rarely or not at all in native proteins, that can be viewed as building blocks for designing different macrocycles. Stepwise residue insertion preserves some of the building blocks and alters others, resulting in a complex propagation of features from the shorter macrocycles to the longer ones.

It was not feasible to characterize each of the 237 macrocycle designs (SEQ ID NOS:1-237) experimentally. Instead we chemically synthesized a subset of 12 peptides (four 7mers: 7.1, 7.2, 7.3, 7.4, two 8mers: 8.1, 8.2, three 9mers: including 9.1, and three 10 mers: including 10.1,10.2), and experimentally characterized their structures by NMR spectroscopy. 10 of the 12 peptides had well-dispersed 1D NMR spectra with the number of backbone HN peaks expected for a single conformation. We collected extensive NOE data (Fig. S11 ) for these peptides, and solved their structures using XPLOR-NIH (18, 19) followed by NOE restrained molecular dynamics (MD) simulations (very similar structures were obtained with an independent large scale enumeration approach). As shown in FIGS. 1-3 and described below, the experimental NMR structures closely matched the design models for 9 of these peptides, and in unrestrained MD simulations, 8 out of these 9 peptides are within 1 Å of the designed structure over 75% of the time.

Unlike proteins, macrocycles cannot be stabilized primarily by the hydrophobic effect as they are too small to form a core that can exclude solvent (20). How then do the sequences of the designs specify their structures? To address this question, we computed the effect on folding of every single substitution to a different amino acid with the same chirality, and to an alanine with opposite chirality, at each position, for all the designs with NMR confirmed structures. For each of the 20*Nres variants full energy landscape calculations were carried out using the large scale backbone enumeration method described earlier (FIGS. 1 and 2 ). These computationally intensive calculations were carried out using cellular phones and tablets of volunteers participating in the Rosetta@Home™ distributed computing project. To evaluate the computed sequence-energy landscape experimentally, we used SLIM (Structures For Lossless Ion Manipulations), an ion mobility mass spectrometry technique that can distinguish different conformations in small molecular structures (21). This technique requires only a small amount of unpurified sample, and enables parallel evaluation of the effects of amino acid substitutions on folding. SLIM results from a set of variants with point mutations of design 7.1 at either the dPro4 or dThr5 position (FIG. 1 ) were consistent with the sequence-energy landscape calculations: the structure was perturbed more by mutations at the dPro4 position than at the dThr5 position, consistent with the computed P_(Near) values. Several general principles emerge from the comprehensive landscape calculations and from folding calculations on permuted sequences. First, L- and D-proline residues play a key role in structure specification: 52% of the positions in which substitutions disrupt the structure are proline residues in the design, and in almost all of the cases, the most destabilizing mutant of a non-proline residue is a substitution to proline. Proline is the most torsionally constrained amino acid, and placement of L- and D-proline residues favors specific turn and kink structures. Second, sidechain-to-backbone hydrogen bonds that either stabilize a structural motif, such as Asp2 in design 8.1, or connect two sides of the structure, such as Glu2 in design 10.1 or Asp3 in design 10.2, are important for structural specification as removal of these interactions substantially reduces the energy gaps. Third, chirality in many cases plays a greater role in structure specification than sidechain identity: replacing an amino acid residue with its mirror is usually more disruptive than changing to a different amino acid with the same chirality. Fourth, for each design, usually fewer than 3 residues (often proline) are critical to defining the fold, leaving the remainder largely free for future functionalization. Even after mutation of the remaining residues to Ala (retaining chirality) a number of the sequences still encode the designed structure. Overall, this global analysis of the effect of substitutions on energy landscape topography defines the sequence determinants of the folding energy landscape in unprecedented detail.

It is instructive to consider these data in the context of the structures and sequences of the individual designs. The 7-residue macrocycles exhibit several recurrent backbone hydrogen bonding patterns, often featuring a proline-nucleated i,i+3/i,i+4 motif (this motif connects residue 1 and three residues after that with a hydrogen bond and connects residue 1 and fourth residue after that (i+4) with a turn). Of the four 7 residue designs experimentally tested, three had structures nearly identical to the design models (FIG. 1 , Table 1), and MD and Rosetta™ calculations on the fourth (design 7.4) suggest it also is close to the design model despite overlap of backbone NH group NOEs. The energy landscape calculations show that the proline nucleating the i,i+3/i,i+4 turn is essential (FIG. 1A,E). The remainder of the structure is largely specified by the designed amino acid chirality with the exception of dPro5 in design 7.2, which packs on the turn-nucleating dPro7. The 8-residue macrocycles are dominated by two major classes, one featuring two i,i+3/i,i+4 motifs, and the other, two criss-cross i,i+3/i+1,i+4 motifs (this motif connects the ith residue and three residues after that (i+3) with a hydrogen bond (I,i+3), and connects residue adjacent to ith residue (i+1) and four residue after residue I (i+4) with a hydrogen bond (I,i+4), resulting in a motif called I,i+3/i+1/i+4) (FIG. 2 , third row). The two 8-residue macrocycles that were experimentally characterized both had NMR structures within 1 Å of the design model. Design 8.1 has multiple slow-exchanging sidechain-sidechain and sidechain-backbone hydrogen bonds, with a structurally critical (FIG. 1A,E) hydrogen bond from Asp2 to the backbone of Thr4, which along with Pro3 stabilizes a sharp kink in the chain. Adjacent to the kink is a BXX (i,i+3/i+1,i+4) motif rare in proteins, anchored by the structurally critical dPro5. Design 8.2 has near-perfect sequence inversion symmetry; the sequence symmetric version of this design with sequence PQrEpqRe and torsion string AAYBXXBY, has half the number of NMR resonances (3 backbone HN instead of 6) consistent with structural S2 symmetry. In contrast to the other 7-8 residue designs characterized, all residues in design 8.2 (SEQ ID NO:234) are important for structure specification (FIG. 1E), with residues involved in multiple sidechain-sidechain hydrogen bonds more essential than the two prolines.

TABLE 1 Different structural features observed for experimentally verified designs. (from top to bottom SEQ ID NOs: 230, 231, 232, 233, 32, 234, 235, 236, and 237) sc- additional number of mediated critical important name sequence turns hbond residues residues design Asp-Thr-dAsn-dPro-Thr-dLys-Asn AA (i, i + 3) Asn7- dPro4 7.1  A   A   Y    Y    A    X   B YAX(i, i + 3/ Thr2(NH) i, i + 4) Asn7- Thr5(C═O) design Asp-Gln-Ser-Glu-dPro-His-dPro BX(i, i + 3) dPro5, 7.2  A   A   B   B    X   B   Y YAA(i, i + 3/ dPro7 i, i + 4) design Gln-Asp-Pro-dPro-Lys-dThr-dAsp XA Pro3 dThr6:  A   B   B    X   A    Y   Y (i, i + 3) polar 7.3 BXA(i, i + 3/ inter- i, i + 4) action with dPro4 design dLys-Tyr-dPro-Glu-Asp-Glu-Arg XA(i, i + 3) Asp5- Asp5 Arg7: 7.4  X    A   X    A   B   A   A AAX(i, i + 3/ Arg(NH) hbond to i, i + 4) Asp5-Arg Asp5 design Asp-Asp-Pro-Thr-dPro-dArg-Gln-dGln YA(i, i + 3) dArg6- Asp2, 8.1  A   B   A   B    X   X    B   Y BXX(i, i + 3/ dGln8(C═O) Pro3, i + 1, i + 4) Gln7- dPro5 dPro5(C═O) Asp2- Thr4(NH) design Pro-Gln-dArg-Gln-dPro-dGln-Arg-dGlu YAA(i, i + 3/ Gln2- most S2 8.2  A   A    Y   B    X    X   B    Y i + 1, i + 4) Arg5- symmetric BXX(i, i + 3/ Gln4 backbone i + 1, i + 4) dArg3- dGln6- Glu8 design Lys-Asp-Leu-dGln-dPro-Pro-Tyr-dHis-Pro AAA(i, i + 3/ dGln4- dPro5, Pro9 9.1  A   A   A    Y    Y   A   A   Y    B i, i + 4) Lys1(C═O) Pro9 stabilizes YAA(i, i + 3/ a bulge i, i + 4) Tyr7  packs against Pro6 design Pro-Glu-Ala-Ala-Arg-dVal-dPro-Arg-dLeu-dThr YA Glu2- long 10.1  A   A   A   A   A    Y    Y   A    Y    Y (i, i + 3) Arg6(NH) range bb YAA(i, i + 3/ Glu2- to bb i + 1, i + 4) Arg6 hydrogen AAA(i, i + 3/ bond i, i + 4) Ala2, dVal6, dLeu9: hydro- phobic packing design Glu-dVal-Asp-Pro-dGlu-dHis-dPro-Asn-dAla-dPro YA Asp3- dVal2, 10.2  A   X    B   B    X    Y    Y   A    Y    Y (i, i + 3) Asn8(NH) dAla8: BX(i, i + 3) hydro- YAX(i, i + 3/ phobic i, i + 4) inter- action

As the macrocycle length increases (9 and 10 residues, FIG. 2 ), so does the entropic cost of folding, and more hydrogen bonds in increasingly diverse patterns are required to stabilize the peptide in the folded state. Three of six experimentally characterized designs had structures close to computational models, one was disordered, and two had well dispersed spectra but the NOE data did not uniquely define the structures. Design 9.1 contains a YAA i,i+3/i,i+4 building block similar to those in the 7-residue macrocycles in which dPro5 plays a critical role (as in the L-Pro/D-Pro in design 7.3, the second proline plays a less critical role). The structure is expanded by insertion of a kink stabilized by Pro9; the remainder of the structure is completed by a tight AAA i,i+3/i,i+4 turn. Design 10.1 contains a 5 residue distorted helix terminated by the critical dPro7. On one face the structurally critical Glu2 in the middle of the helix makes a long range sidechain-backbone hydrogen bond to Arg8, and on the other, Ala3, dVa16, and dLeu9 form a non-polar cluster. Design 10.2 contains BX, YA and the rare YAA building blocks each beginning with a proline residue; of these, Pro4 in the BX motif is the most critical. As with 10.1, the building blocks are held together by nonpolar interactions (between dVal2 and dAla8) on one face, and a long-range sidechain-backbone hydrogen bond (from Asp3 to Asn8) on the other; both dVal2 and Asp3 are critical for specifying the structure.

The entropic cost of folding continues to increase with increasing number of residues, and for 11-14 residue macrocycles, additional crosslinks to form bicyclic structures were required to obtain single states amenable to NMR structure determination. We solved the structures of 3 such designs (FIG. 2 , row IV) that feature long-range backbone-backbone hydrogen bonds. Design 11_SS has a i,i+1/i+1,i+4 building block (this motif connects residue 1 and one residues after that with a hydrogen bond (0+1), and also connects the residue after residue 1 (i+1) with and fourth residue after that (i+4) with a turn) with a critical proline in the first position preceded by a cysteine that forms a critical disulfide to a cysteine preceding a YA turn. Design 12_SS has a rare BXAX i,i+4/i,i+5 turn (this motif connects residue 1 and four residues after that with a hydrogen bond (I,i+4) and connects residue 1 and fifth residue after that (i+5) with a hydrogen bond (I,i+5)), which exhibits higher flexibility in NMR structure, and a disulfide between backbone hydrogen bonding residues. The more compact and complex 14_SS design has a network of interleaved local and non-local backbone hydrogen bonds (22), and a D-Cys to L-Cys disulfide bond.

The wide variety of shapes spanned by our macrocycle designs, together with their high stability and high predicted tolerance for sequence mutations, makes them attractive starting points for developing new therapeutics. One approach to inhibitor design is scaffolding loops at binding interfaces in the PDB; such scaffolding can increase binding affinity by pre-organizing the loops in the binding-competent conformation, enable additional interactions with the target, and improve cell permeability and oral bioavailability (23). We found that 907 of the 1017 “hot loops” identified at protein-protein interfaces by Kritzer and coworkers (24) could be scaffolded by one or more of our designs.

The finding that 70% of the experimentally-tested 7- to 10-residue macrocycle designs adopt single unique structures close to the computationally-designed models suggests that most of the 200+ new macrocycle designs with high computed Boltzmann weights fold as designed, increasing the known repertoire of possible macrocycle structures by more than two orders of magnitude. Our results demonstrate that the principles and energy functions developed in recent years to design proteins have quite broad applicability, transferring over to much smaller systems even though (1) the factors dominating the folding of proteins (for example, the hydrophobic effect) differ considerably from those that stabilize conformations of small peptide macrocycles (local hydrogen bonding patterns and intrinsic conformational preferences of amino acid building-blocks), and (2) all designed proteins to-date contain regular α-helix or β-sheet structures, while small peptide macrocycles lack these and instead contain a wide range of local structures some of which are rarely or never observed in proteins.

There are two clear paths forward for engineering new macrocyclic therapeutics by exploiting the rigidity and stability of the designs together with the freedom to choose the identities of the non-structure specifying positions. The first is experimental: libraries can be constructed in which at each position all residues compatible with the structure are allowed (identified as described above using large-scale energy landscape calculations), and screened for target binding using current library selection methodologies. The second is computational: each macrocycle can be docked against the target (using for example rigid body docking or “hot loop” superposition), and the interface residues designed to maximize binding affinity. Unnatural amino acids can be incorporated in either approach, but the second has the advantage that new functionalities—such as known active site binding groups—can be strategically placed to maximize binding affinity. Beyond binding, the control over geometry and chemistry provided by our approach should contribute to understanding the structural correlates of membrane permeability and other desirable pharmacological properties.

Methods Backbone Conformational Sampling

Conformations of 7- to 14-residue polyglycine backbones were sampled using the previously-described Rosetta™ simple_cycpep_prediction application (15), with key modifications. Unlike the Rosetta™ ab initio method used for protein structure prediction (25), simple_cycpep_predict does not make use of fragments of proteins of known structure, since such fragments poorly cover the conformational space accessible to chains of mixtures of L- and D-amino acids. Instead, it uses an efficient kinematic closure-based algorithm (17, 26) that samples only closed conformations to limit the search space. Briefly, the sampling process consisted of the following steps: first, a linear chain of glycine residues was constructed, one residue of which was selected randomly to be the “anchor” residue for subsequent loop closure steps. The N- and C-terminal residues were excluded from being the anchor residue. This residue's mainchain φ and Ψ dihedral values were drawn randomly from a flat, symmetric Ramachandran distribution based on the glycine Ramachandran map. Second, a bond was declared between the nitrogen of the N-terminal residue and the carbonyl carbon of the C-terminal residue, and the Rosetta generalized kinematic closure (GenKIC) module was invoked to close the loop consisting of all residues but the anchor residue. During this process, the φ and Ψ′ dihedral values of all but three residues in the loop were randomized, biased by the same flat, symmetric distribution used to randomize the anchor residue, and the φ and Ψ′ dihedral values for the remaining three residues were determined algebraically to ensure loop closure with ideal peptide bond geometry at the cutpoint (the bond between the first and last residues). In preliminary design calculations, we found that unique low-energy structures with energy gaps greater than ˜10 k_(B)T (˜6 kcal/mol) could only be obtained for macrocycles containing at least N/3 backbone hydrogen bonds; therefore, in subsequent sampling calculations, of the many closure solutions found, those with mainchain hydrogen bond counts below the threshold value were discarded. Third, the cyclic backbone was relaxed with the Rosetta FastRelax™ protocol (27) using the all-atom Rosetta™ energy function “ref2015” (28, 29), with the rama_prepro and p_aa_pp mainchain potentials made symmetric, as described previously (15). Up to 108 samples were attempted, not all of which yielded closed solutions with the desired minimum number of hydrogen bonds.

Sampling was carried out on the “Mira” Blue Gene/Q supercomputer (Argonne labs) or Amazon Web Service (AWS). For efficiency, a new multi-level hierarchical job distribution and data reduction scheme was implemented for use on massively parallel architecture. In performance benchmarks, this yielded linear performance scaling up to at least 250,000 CPUs.

Energy-Based Clustering and Data Reduction

The sampling described above yields up to millions of backbones, making the problem of identifying repeatedly-sampled conformations a difficult problem in data reduction. While many algorithms for clustering large datasets have been developed (30-32), this particular problem has an interesting feature: Rosetta's energy calculations can be used to establish a rank order for the degree to which elements in the dataset are “interesting”, providing a useful means of selecting cluster centers without performing a prohibitively expensive all-to-all RMSD calculation.

We developed a simple energy-based clustering algorithm for this problem: first, the energy of each input structure is scored using the Rosetta™ all-atom energy function (ref2015), and minimal backbone information for every structure is stored in an unclustered pool. Second, the lowest-energy structure in the unclustered pool is selected as the center of the first cluster. This structure is moved from the unclustered pool into the first cluster, and the backbone RMSD between this structure and every circular permutation of every structure remaining in the unclustered pool is calculated. Those structures for which at least one circular permutation lies within a threshold RMSD from the current cluster center are also removed from the unclustered pool and added to the new cluster. For our purposes, we typically used an RMSD threshold of 1.25 Å. Third, the lowest-energy structure remaining in the unclustered pool is selected as the center of the next cluster, and the second step is repeated. This process continues for subsequent clusters until no structures remain in the unclustered pool. Note that, unlike Voronoi clustering schemes, this “cookie-cutter” approach deliberately gives precedence to lower-energy clusters. Although simple, we found that this approach worked well for our large datasets, yielding lower-energy clusters that were particularly easy to stabilize with suitable amino acid sequences.

Torsion Bin-Based Clustering

We developed a custom PyRosetta™ Python script for re-clustering the cluster centers from the previous, RMSD-based clustering step. Briefly, this script assigns a torsion bin string to each input structure, sorting all circular permutations in both chiralities of the bin string alphabetically and selecting the first in order to allow structures with different circular permutations to be compared easily. A string representing a hydrogen bonding pattern is also assigned to each input structure, circularly permuted to match the circular permutation of the torsion bin string. The structure is then assigned to a cluster with the same torsion bin string and hydrogen bonding pattern, or, if no such cluster has yet been encountered, a new cluster is created and the structure is assigned to that new cluster. The process is repeated until all input structures have been assigned to clusters.

Computational Sequence Design

The Rosetta FastDesign™ module was used for sequence design. FastDesign™ performs alternating rounds of side-chain identity and rotamer optimization (using the Rosetta Packer™ module) and torsion-space energy minimization (using the Rosetta Minimizer™ module), with the repulsive term of the Rosetta™ energy function, fa_rep, ramped from 2% of its normal value to 100% of its normal value from round to round.

FastDesign™ seeks to minimize the energy of a designed structure. However, there were additional requirements that we wished to impose during the design process. Some such requirements were intended to limit the conformational flexibility of the designs produced, and to maximize the chances of the designed structure representing a unique low-energy conformation. To this end, we wished to require a minimum L- or D-proline content, for example. Other requirements were practical needs for synthesis (e.g. the need for at least one L-aspartate or L-glutamate in the sequence to allow resin tethering during cyclization), or for characterization (e.g. the need for at least one positively-charged residue to facilitate mass spectrometry).

To this end, we implemented a non-pairwise-decomposable term, called aa composition, which allowed users to define a nonlinearly-ramping penalty for deviation from a desired amino acid composition to guide the Packer™ to find sequences with desired compositions. This allowed us to require a minimum proline count, and at least one L-aspartate or L-glutamate and one positively charged residue per design.

We also implemented two new residue selectors, called the PhiSelector™ and BinSelector™, to provide additional control over the Packer™. We used these to require that the Packer™ consider only L-amino acid residues at positions with a mainchain φ value less than zero, and only D-amino acid residues at positions with a mainchain φ value greater than zero.

During early design runs, we found that Rosetta™'s normally pairwise-decomposable scoring function would erroneously favor structures in which more than two hydrogen bond donors made bonds to a single acceptor. Since it is difficult to change the hydrogen bonding architecture to give favorable scores to a maximum of two donors binding to an oxygen acceptor (since such scoring would necessarily be non-pairwise-decomposable), we instead implemented a filter to discard designs with this pathology.

Computational Validation: Energy Landscape Analysis of Designed Macrocycles and their Mutants

For each torsion bin string and hydrogen bonding pattern, the lowest-energy sequence designed was picked as a representative of that cluster. A subset of such low-energy structures (from 44% of all designs for length 7 to 3% of all designs for length 10) was subjected to a final round of computational validation using the simple_cycpep_predict application, as described previously (15). As for the sampling of polyglycine conformations, large numbers of backbone conformations were sampled for each sequence tested, but this time, the sampling was biased based on the Ramachandran map of each amino acid residue in the sequence. Each sample was subjected to full side-chain rotamer optimization and energy minimization using the Rosetta FastRelax™ protocol (27). The “foldability” of each macrocycle was evaluated based on the estimated fractional occupancy of the native state (a value that we call P_(Near)), and on the energy gap between the native structures and other low energy models, as reported previously (15). A P_(Near) value of >0.9 and energy gap of <−0.1 was selected as the basic threshold for acceptance. Additionally, the plot of energy vs. RMSD was then visually inspected.

For a subset of macrocycles, large-scale landscape analysis was performed. Each residue in the initial sequence was systematically mutated to the other 18 amino acid residues of the same chirality, and to alanine with mirror chirality, in the input sequences provided to the simple_cycpep_predict application. These large scale computational analyses of the energy landscape was performed using the Berkeley Open Infrastructure for Network Computing (BOINC) as part of the Rosetta@Home™ project, mostly using volunteer cellular telephones as the computing hardware (though some earlier predictions were carried out using volunteer desktop computers, or using the Argonne “Mira” Blue Gene/Q system used for poly-Gly conformational sampling).

Scrambled sequences were generated by randomly assigning residues to different positions in the structure.

After generation of results, site-saturation mutagenesis plots were generated based on P_(Near) values (see equation below for P_(Near)) for each structure, with λ set to 1 Å and a value of k_(B)T of 0.62 kcal/mol (equivalent to 37° C.). For two of the macrocycles, different combinations of 2 and k_(B)T (0.5, 0.75, 1, 1.5 for 1 and 0.5, 0.75, 1, 2 for k_(B)T) were tested and the value with more dynamic range (i.e. values that showed the difference between a high-quality vs. low-quality energy funnel best) were selected. Double mutants were generated and analyzed using similar methods described above.

Equation 1: Definition of P_(Near), a measure of the quality of an energy function. P_(near) approximates the Boltzmann-weighted probability of finding the structure in a conformation near the native conformation.

$P_{near} = \frac{\sum_{i = 1}^{N}{{\exp\left( {- \frac{{rmsd}_{i}^{2}}{\lambda^{2}}} \right)}{\exp\left( {- \frac{E_{i}}{k_{B}T}} \right)}}}{\sum_{j = 1}^{N}{\exp\left( {- \frac{E_{j}}{k_{B}T}} \right)}}$

Turn Type Analysis and Measurement of RMSD to Hot Loops

We defined a turn as a semi-independent part of a macrocycle structure that is connected internally through backbone-to-backbone hydrogen bonds, but which lacks hydrogen bonds to other parts of the structure. For each structure, different turn types were defined by their torsion bin strings and hydrogen bond patterns. Similar analysis was performed on a subset of structures from the PDB, and the frequencies were then calculated and compared. The redundancy of the PDB subset was reduced to 30%—that is, no two PDB chains in the set had more than 30% sequence identity.

From all the hot loops generated by Kritzer and coworkers (24), those that contained continuous stretches of amino acids were selected. Each loop, and small truncations of it (one residue shorter from each side) were then compared to a library of macrocycles that passed computational consistency check. For every motif and scaffold, a matrix of pairwise distances between C-alpha atoms and a vector of dihedral angles for every four consecutive C-alpha atoms was computed. For every possible alignment of linear motif to cyclic scaffold, Root-Mean-Square of the differences of both the distance matrices (distance RMS) and the dihedral vectors (dihedral RMS) is reported. Macrocycles that at least in one position had a distance RMSD of less than 1 Å and a dihedral RMSD of less than 10 degrees (i.e. contained a portion matching the motif backbone) were considered to be plausible stabilizing scaffolds for the given motif. A complete list of these hot loops and the results are available as a supplementary file.

Synthesis, Purification, and Mass Spectrometry of Macrocycles

All peptides were synthesized using standard Fmoc solid phase peptide synthesis (SPPS) on preloaded and sidechain-linked Fmoc-Asp(Wang resin LL)-ODmab or Fmoc-Glu(Wang resin LL)-ODmab resin. Linear, protected peptides were built on a CEM Liberty Blue Peptide Synthesizer with microwave heating at coupling and deprotection steps. After the final Fmoc deprotection, the resin was treated with 2% (v/v) hydrazine monohydrate in dimethylformamide (DMF) to remove the C-terminal Dinah protecting group; the N- and C-termini were then joined on-resin by a coupling reaction. A cleavage cocktail of TFA:Water:TIPS:DODT (92.5:2.5:2.5:2.5) used for global deprotection of side-chains and to cleave the peptide from the resin. After the removal of residual TFA by evaporation, peptides were ether precipitated and further purified using RP-HPLC.

Crude peptides were purified using an Agilent Infinity Preparative HPLC with an Agilent Zorhax™ SB-C18 column (9.4 mm×250 mm). A linear gradient of 1%/min for Solvent B (ACN with 0.1% TFA) and flow rate of 5 ml/min was used for purification to collect fractions with pure peptides. Mass and purity of peptides were confirmed using electrospray ionization mass spectrometry (ESI-MS) on a Thermo Scientific TSQ Quantum Access mass spectrometer.

For disulfide-stapled peptides, cyclic reduced peptides were air-oxidized in 0.1 M ammonium bicarbonate buffer (pH 8.3) for 48 hours, and purified again using RP-HPLC. Some of the disulfide-containing peptides were synthesized with Fmoc-Cys(Acm)-OH at the cysteine positions. Following synthesis and cyclization, the resin was treated with 8 eqs. of iodine in 4:1 DMF:methanol overnight to remove the Acm protecting groups and facilitate disulfide bond formation. After iodine treatment, the resin was washed with 2% w/v ascorbic acid in DMF, rinsed with dichloromethane (DCM) and cleaved and purified as normal.

Nuclear Magnetic Resonance (NMR) Spectroscopy Studies of the Designed Macrocycles

Each peptide macrocycle was dissolved at concentrations of ˜5 mg/mL at a pH between 3.0 and 5.5 in 10% D20, with up to 5% glycerol-ds added. All NMR data were collected on a DRX 500 MHz, an Avance™ III 600 MHz, or an Avance™ III 800 MHz spectrometer, equipped with TCI cryoprobe and triple-axis gradient (Bruker). Unless otherwise noted, all NMR data were collected at 5° C. and 25° C. using pulse sequences with excitation sculpting water suppression. Data were processed with TOPSPIN™ v. 3.5 (Bruker) or NMRPIPE™ (34) and visualized with Sparky. Initial screening of designed cyclic peptides for discrete structure involved recording 1D ¹H spectra at 25° C. and selecting peptides with sharp, and well dispersed backbone amide resonances. The small size of the peptides (<=14 residues) selected for structural analysis allowed for complete proton backbone and side chain resonance assignment using 2D [¹H,¹H] TOCSY; including many stereospecific assignments. To facilitate quantitative evaluation of internuclear distances, sample temperatures were dropped to 5° C. and both 2D [¹H,¹H]-ROESY with a 200 ms mixing time and 2D [¹H,¹H] NOESY spectra were collected using mixing times of 100 ms and 500 ms. For designs 8.1 and 14_SS a full NOESY buildup curve (50-75 ms mixing time) was collected to ensure linear behavior of the glycerol containing samples of small peptides S28). Because it is currently not economical to prepare uniformly ¹³C and ¹⁵N-labelled peptides using solid phase methods, and because natural abundance experiments are resource-intensive, only a set of ¹⁵N assignments were measured using natural abundance 2D [¹⁵N,¹H] SOFAST HMQC for designs 7.1, 7.2. 8.1, 8.2. 12_SS, 14_SS. For longer peptide designs or designs with clear overlap in the 2D [¹H,¹H] TOCSY we also collected natural abundance 2D [¹³C,¹H] HMQC.

Nuclear Overhauser Effect (NOE) Constraint Consistency Check

To evaluate whether NOE constraints alone can predict the designed structure, we first used Rosetta to relax 5 macrocycles from the Protein Data Bank (PDB) and Cambridge Structural Database (CSD) that shared the same criteria as our peptides (4ME6 from the PDB and CUQYUI, DUYTIA, MANGO, and UZUKUW from the CSD); this was repeated 20 times. Based on the observed distribution of energies after relaxation, we set the following filters for the score terms below and selected structures that passed these filters from our previous landscape analysis:

-   -   omega=1, fa_rep=10, fa_intra_rep=0.5, pro_close=5, rama_prepro=3

Each structure was then rescored, using Rosetta, based solely on how well it satisfied the NOE constraints and the scores vs. RMSD to design were plotted.

NMR Structure Determination of Designed Macrocycles

A set of 200 structures were calculated for well-behaved designs with the Xplor-NIH software package using torsion angle dynamics and simulated annealing. Initial folding was conducted from a single starting template of randomized torsional angles for the cyclic peptide after patching L- or D-stereoisomers. Distance restraints were derived from NOE intensities at 100 and 500 ms mixing times in 2D [¹H,¹H] NOESY spectra recorded at 500 or S00 MHz and were sorted into Strong (2.5 0.7 0.7), Medium (3.5 1.5 1.5) and Weak (4.5 2.0 2.0) bins based on relative peak intensities to aromatic resonance signals. A soft square potential was used for NOE restraints for initial folding and convergence was established when there were no NOE violations greater than 0.5 Å of the calculated structures.

After initial folding, hydrogen-bonding restraints were inferred from proximal atoms, identified by cross-strand or nearest neighbor amide NOE cross peaks in the 2D [¹H-¹H] NOESY or monitoring slow exchanging protons with ID ¹H CLEANEX-PM pulse sequences (mixing time 0-500 ms). After backbone hydrogen bonding was established, structures were re-calculated as described incorporating hydrogen bonds as NOE restraints using a biharmonic potential. Throughout folding and refinement, only NOE and van der Waals terms were active during structure calculation. Due to lack of uniform labeling and peak overlap we were not able to make clear predictions of backbone dihedral angle restraints or coupling constants. The torsional database constraints were also left unrestrained due to lack of sufficient information for handling D-amino acids. To refine the structures based on NMR experiments, we launched MD simulations with NOE constraints. In particular, for each structure, a simulated annealing from 350K to 310K followed by a 10-ns production run was performed (35). For each atom pair measured by NOE, a distance restraint (k=1000 nm⁻¹) was applied throughout the simulation. The 20 conformations with the lowest total energy were selected for further analysis.

MD Simulation of Designed Macrocycles

Molecular Dynamics simulations were performed using GROMACS™ 2016.1(36, 37) with the Amber™ 99SB-ILDN forcefield (38). Each peptide was solvated in a dodecahedron box of explicit TIP3P waters(39) and neutralized with either sodium or chloride ions. The solvated systems were energy-minimized using the steepest descent minimization method. Next, the system was equilibrated for 1 ns under the NPT ensemble with position restraints (1000 kJ mol⁻¹ nm⁻¹) applied on all the heavy atoms of the peptide. During this equilibration, pressure coupling to 1 atm was performed with the Berendsen barostat (40), and temperature coupling to 310 K using the velocity-rescaling thermostat (41). From each equilibrated system, 10 simulations of 100 ns were performed in the NVT ensemble. The systems were simulated using periodic boundary conditions. A cutoff at 10 Å was used for van der Waals and short-range electrostatic interactions. The Particle-Mesh Ewald (PME) summation method was used for the long-range electrostatic interactions (42). The Verlet cut-off scheme was used(43). All chemical bonds were constrained using the LINCS algorithm (44). The integration time-step was 2 fs, and simulations were analyzed using GROMACS tools. We calculated the root-mean-square deviation (RMSD) of the position of the Ca atoms of the peptides, compared to the initial conformation, using gmx rms. The peptides were aligned to the C_(α) of the initial conformation. The Ramachandran plots were calculated using gmx chi, and plotted using the Matplotlib histogram2d function.

For two of the structures, design 8.1 and 10.1, we also performed our analysis for the mirrored structure of the designs to make sure that our calculations are not energetically biased against L- or D-amino acids. As shown in fig. S29, the results are comparable; thus, we only performed simulations of the designed structure (and not its mirror image) for the rest of the macrocycles.

For designs 7.3 and 7.4, we performed long (>1 μs) molecular dynamics simulations to analyze the dynamics of folding and different conformations explored by the macrocycles. The Markov state model that captures movement of the macrocycle was generated by MSM builder, and the dynamics of movement were described using a time-structure independent component analysis (t1CA) model(44-47).

Ion Mobility Spectrometry Analysis

The single-site mutant libraries of design 7.1 were synthesized with a process similar to that described above with an additional step. For the residues for which the mutation was made (dPro4 or Thr5), the resin was removed from the synthesizer and split into 6 pools. Each pool had its respective amino acid coupled individually using the synthesizer (D-Pro, D-Ser, D-Asn, D-Asp, D-Met, D-Arg for position 4 and Thr, Ser, Leu, Gln, Glu, Trp for position 5). After all pools of resin were loaded with the desired amino acid they were recombined and the remaining amino acids in the sequence were coupled as normal. Cleavage of the resin was performed using the same cleavage cocktail described above. All expected species were confirmed by mass spectrometry.

All samples were prepared in 50% aqueous methanol acidified with 0.1% formic acid. The solutions were infused at an infusion rate of 300 nL/min and electrosprayed in the positive mode using an etched emitter (20 mm i.d.). The formed ions were transmitted through a heated inlet capillary (130° C.) into a high-resolution Structures for Lossless Ion Manipulations Ion Mobility Mass Spectrometer (SLIM IM-MS) platform for high resolution ion mobility spectrometry(21). Ions were accumulated in an ion funnel trap(48) for 2 ms and then released to SLIM IM-MS. The SLIM module was similar to that of the SLIM serpentine design previously reported(49, 50), but has a path length of 15.9 m that allows for multiple passes through the serpentine path for higher ion mobility spectrometry resolution. The SLIM module was integrated with an Agilent 6224 TOF MS equipped with a 1.5 m extended flight tube via a rear ion funnel and RF-only quadrupole. All SLIM separations were performed at ˜2.5 Torr N2 with the following parameters: wave speed of 160 m/s, wave amplitudes of 40 V, guard electrode voltage of 6 V, and RF frequency of 1.0 MHz and amplitude of 380 V_(p-p). Data were acquired on an 8-bit ADC (analog-to-digital converter) using a control software developed in-house.

Protease Assay:

Protease assay was performed using PRONASE® Protease derived from Streptomyces griseus from EMD Millipore (product #53702). 0.2 μmole of each peptide tested was added to 200 μl of 50 mM ammonium acetate buffer, pH 8, supplemented with 0.01 M calcium acetate. 5 μl of this starting material was mixed with μl TFA and kept as the time 0 sample. To this mixture we added 2 μl of 2 mg/ml protease mix stock (prepared by dissolving in water) and incubated at 37° C. At different time points, 5 μl of the reaction mixture was taken out and quenched by addition of 5 μl TFA. To track protease cleavage, each sample was analyzed by LC/MS (Thermo Scientific Accela HPLC system connected to Thermo Scientific TSQ Quantum Access mass spectrometer) using an Agilent ZORBAX™ StableBond™ 300 C18, 4.6×150 mm, 5 μm as the chromatography column.

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1. A macrocyclic polypeptide comprising or consisting of a polypeptide having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids.
 2. The polypeptide of claim 1, wherein the polypeptide has at least one proline residue.
 3. The polypeptide of claim 1, wherein the polypeptide is between 7 and 14 amino acid residues in length, or between 7 and 10 amino acid residues in length.
 4. The polypeptide of claim 2, wherein each amino acid substitution occurs at a non-proline position.
 5. The polypeptide of claim 1, wherein the amino acid substitutions do not include any non-proline residues being substituted with proline.
 6. The polypeptide of claim 1, wherein each amino acid substitution maintains the chirality of the amino acid replaced.
 7. The polypeptide of claim 1, wherein each amino acid substitution is an alpha amino acid.
 8. The polypeptide of claim 1, wherein the polypeptide has at least 2, 3, 4, 5, 6, 7, 8, or more D amino acid residues.
 9. The polypeptide of claim 1, wherein the polypeptide has no more than 2 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof.
 10. The polypeptide of claim 1, wherein the polypeptide has no more than 1 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof.
 11. The polypeptide of claim 1, comprising or consisting the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof.
 12. A polypeptide library, comprising two or more polypeptides having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids.
 13. The polypeptide library of claim 13, comprising ten or more polypeptides having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids.
 14. The polypeptide library of claim 13, comprising fifty or more polypeptides having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids.
 15. The polypeptide library of claim 14, comprising two hundred or more polypeptides having no more than 3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NO: 1-237 or a mirror image thereof, wherein the polypeptide includes both L and D amino acids.
 16. Use of the polypeptide of claim 1 as a scaffold for target-based drug design or to screen molecules of interest for binding to one or more of the polypeptides. 